The overall objectives of this proposal are to better understand the regulation of insulin secretion by free calcium ions (Ca2+) and the role of abnormal Ca2+ signaling in the pathophysiology of Type 2 diabetes mellitus (T2DM). Glucose stimulation evokes changes in intracellular Ca2+ ([Ca2+]c) in islets and insulin- secreting cell lines that temporally correlate with insulin secretion. Although much is known about the contribution of Ca2+ influx through voltage-gated Ca2+ channels to B-cell insulin secretion, our knowledge of the contribution of endoplasmic reticulum (ER) Ca2+ stores is limited. This is in part due to few reports of direct measurements of secretagogue effects on [Ca2+]ER and incomplete understanding of the mechanisms that regulate ER Ca2+ stores in B-cells. The proposed experiments will focus on defining these mechanisms. Preliminary studies suggest that sarcoendoplasmic reticulum Ca2+-ATPase (SERCA), a key regulator of ER Ca2+ homeostasis, is impaired in islets from the db/db mouse model of T2DM. This raises the intriguing possibility that loss of B-cell function in T2DM is related to defects in ER Ca2+ signaling. We will employ a combination of biosensor technology, RNA silencing, novel transgenic and imaging approaches to identify and characterize underlying regulatory mechanisms. The proposed experiments will [1] identify spatial and temporal interplay between cytoplasmic and ER Ca2+ signaling in single cells; [2] determine kinetics of signals that affect ER Ca2+ mobilization; [3] define novel mechanisms that regulate ER Ca2+ store refilling; [4] define the role of mitochondria in regulation of ER Ca2+ homeostasis; [5] determine whether stromal interaction molecule (STIM) couples ER Ca2+ levels with store-operated Ca2+ entry (SOCE); and [6] define the role of ER Ca2+ signaling defects in B-cell dysfunction associated with diabetes. The following hypotheses will be tested: [1] glucose stimulates ER Ca2+ signaling; [2] glucose-induced [Ca2+]c and [Ca2+]ER oscillations are temporally and causally interrelated; [3] STIM1 is expressed in B-cells and essential for SOCE; [4] mitochondria and plasma membrane-related Ca2+-ATPase-1 (Pmr-1) regulate ER Ca2+ homeostasis; [5] B-cell dysfunction in T2DM is due to defects in ER Ca2+ homeostasis consequent to decreased expression of SERCA and STIM. We will test these hypotheses in MIN6 B-cells, islets and primary B-cells from transgenic biosensor mice and db/db mice. Identification and characterization of the signaling pathways that control ER Ca2+ is a necessary step in advancing our knowledge of the molecular mechanisms regulating B-cell Ca2+ signal transduction and function. The experiments will contribute new information essential to improve understanding of the pathogenesis of T2DM, and facilitate development of novel strategies used in the preservation and maintenance of B-cell function in patients with diabetes. [unreadable] [unreadable] [unreadable]